Structure of the human mitochondrial monoamine oxidase B: new chemical implications for neuroprotectant drug design.

Monoamine oxidase B (MAO-B) is an outer mitochondrial membrane-bound flavoenzyme that is a well-known target for antidepressant and neuroprotective drugs. The 3A resolution structure of recombinant human MAO-B originally determined was of the enzyme complexed with pargyline, an irreversible inhibitor covalently bound to the N5 atom of the flavin coenzyme. The crystal structure shows that the enzyme is dimeric. Each monomer binds to the membrane via a C-terminal transmembrane helix and by apolar loops located at various positions in the sequence. Substrate binding to the enzyme involves negotiating a loop covering a 290A3 entrance apolar cavity before reaching an apolar 420A3 substrate cavity where the flavin coenzyme is located. The 1.7A isatin-MAO-B structure allowed a detailed examination of the enzyme's active site. A novel specific reversible MAO-B inhibitor, which is found as a contaminant in polystyrene plastics (1,4-diphenyl-2-butene), binds in both the entrance and the substrate cavity. Analogous MAO-B-specific inhibitors that bind in a manner traversing both cavities include trans-trans farnesol and chlorostyrylcaffeine. The rotation of the Ile199 side chain to an "open" conformation plays an essential role in this specificity. These results form a molecular basis for the design of new human MAO-B-specific reversible inhibitors.

A2A antagonist prevents dopamine agonist-induced motor complications in animal models of Parkinson's disease.

Adenosine A(2A) receptors, abundantly expressed on striatal medium spiny neurons, appear to activate signaling cascades implicated in the regulation of coexpressed ionotropic glutamatergic receptors. To evaluate the contribution of adenosinergic mechanisms to the pathogenesis of the response alterations induced by dopaminergic treatment, we studied the ability of the selective adenosine A(2A) receptor antagonist KW-6002 to prevent as well as palliate these syndromes in rodent and primate models of Parkinson's disease. In rats, KW-6002 reversed the shortened motor response produced by chronic levodopa treatment while reducing levodopa-induced hyperphosphorylation at S845 residues on AMPA receptor GluR1 subunits. In primates, KW-6002 evidenced modest antiparkinsonian activity when given alone. Once-daily coadministration of KW-6002 with apomorphine prevented the development of dyskinesias, which appeared in control animals 7-10 days after initiating apomorphine treatment. Animals initially given apomorphine plus KW-6002 for 3 weeks did not begin to manifest apomorphine-induced dyskinesias until 10-12 days after discontinuing the A(2A) antagonist. These results suggest that KW-6002 can attenuate the induction as well as the expression of motor response alterations to chronic dopaminergic stimulation in parkinsonian animals, possibly by blocking A(2A) receptor-stimulated signaling pathways. Our findings strengthen the rationale for developing A(2A) antagonists as an early treatment strategy for Parkinson's disease.

Inhibition of human MAO-A and MAO-B by a compound isolated from flue-cured tobacco leaves and its neuroprotective properties in the MPTP mouse model of neurodegeneration.

Prompted by the findings that smokers have lowered brain and blood platelet monoamine oxidase-A and -B activities compared to non-smokers and that smokers have a lowered incidence of Parkinson's disease, we have examined the neuroprotective properties of an MAO inhibitor, 2,3,6-trimethyl-1,4-naphthoquinone (TMN), which is present in the tobacco plant and smoke in the MPTP C57BL/6 mouse model of neurodegeneration. Dopamine (DA) levels in the striata of mice treated with TMN prior to the administration of MPTP were significantly higher than DA levels in the striata of mice receiving MPTP only, thus indicating a degree of neuroprotection in this model of Parkinson's disease. The potential consequences on MAO activity of long term exposure to this compound need to be evaluated. Furthermore, there is evidence for the presence of other inhibitors in the tobacco leaf and smoke, including compounds with irreversible MAO inhibitory properties. Although there is no evidence to link the lowered activities of MAO to the lowered incidence of Parkinson's disease in smokers, the neuroprotective effects of TMN in the MPTP mouse model suggest that such a relationship is worthy of further evaluation.

Rescue of dying neurons by (R)-deprenyl in the MPTP-mouse model of Parkinson's disease does not include restoration of neostriatal dopamine.

Chronic (8- to 10-week) administration of the selective, potent, and irreversible monoamine oxidase B inhibitor (R)-deprenyl has been shown to increase the tyrosine hydroxylase immunoreactivity in the substantia nigra of mice that had been treated three days earlier with a neurotoxic dose of the parkinsonian-inducing agent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). This reported rescuing of lesioned nigrostriatal cell bodies by (R)-deprenyl prompted us to investigate if this (R)-deprenyl treatment also could restore neostriatal dopamine levels that are depleted by MPTP. The results of these experiments show that long term (8 or 10 weeks) treatment with (R)-deprenyl beginning three days post MPTP administration did not result in restoration of depleted neostriatal dopamine levels in C57BL/6 mice. We conclude that, although (R)-deprenyl may rescue MPTP-injured nigrostriatal neurons, it does not lead to functional recovery of these neurons as measured by the restoration of neostriatal dopamine levels.

Rat liver microsomal enzyme catalyzed oxidation of 4-phenyl-trans-1-(2-phenylcyclopropyl)-1,2,3,6-tetrahydropyridine.

As part of our ongoing studies to characterize the catalytic pathway(s) for the monoamine oxidase and cytochrome P450 catalyzed oxidations of 1,4-disubstituted 1,2,3,6-tetrahydropyridinyl derivatives, we have examined the metabolic fate of 4-phenyl-trans-1-(2-phenylcyclopropyl)-1,2,3,6-tetrahydropyridine in NADPH supplemented rat liver microsomes. Three metabolic pathways have been identified: (1) allylic ring alpha-carbon oxidation to yield the dihydropyridinium species, (2) nitrogen oxidation to yield the N-oxide and (3) N-dealkylation to yield 4-phenyl-1,2,3,6-tetrahydropyridine and cinnamaldehyde. A possible mechanism to account for the formation of cinnamaldehye involves an initial single electron transfer from the nitrogen lone pair to the iron oxo system Fe(+3)(O) to form the corresponding cyclopropylaminyl radical cation that will be processed further to the final products. The reaction pathway leading to the dihydropyridinium metabolite may also proceed via the same radical cation intermediate but direct experimental evidence to this effect remains to be obtained.

Neuroprotection in the MPTP Parkinsonian C57BL/6 mouse model by a compound isolated from tobacco.

Epidemiological evidence suggests a lower incidence of Parkinson's disease in smokers than in nonsmokers. This evidence, together with the lower levels of brain monoamine oxidase (MAO) activity in smokers and the potential neuroprotective properties of MAO inhibitors, prompted studies which led to the isolation and characterization of 2,3,6-trimethyl-1,4-naphthoquinone (TMN), an MAO-A and MAO-B inhibitor which is present in tobacco and tobacco smoke. Results of experiments reported here provide evidence that this compound protects against the MPTP-mediated depletion of neostriatal dopamine levels in the C57BL/6 mouse. These results support the hypothesis that the inhibition of MAO by constituents of tobacco smoke may be related to the decreased incidence of Parkinson's disease in smokers.

Neuroprotection by caffeine and A(2A) adenosine receptor inactivation in a model of Parkinson's disease.

Recent epidemiological studies have established an association between the common consumption of coffee or other caffeinated beverages and a reduced risk of developing Parkinson's disease (PD). To explore the possibility that caffeine helps prevent the dopaminergic deficits characteristic of PD, we investigated the effects of caffeine and the adenosine receptor subtypes through which it may act in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxin model of PD. Caffeine, at doses comparable to those of typical human exposure, attenuated MPTP-induced loss of striatal dopamine and dopamine transporter binding sites. The effects of caffeine were mimicked by several A(2A) antagonists (7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH 58261), 3,7-dimethyl-1-propargylxanthine, and (E)-1,3-diethyl-8 (KW-6002)-(3,4-dimethoxystyryl)-7-methyl-3,7-dihydro-1H-purine-2,6-dione) (KW-6002) and by genetic inactivation of the A(2A) receptor, but not by A(1) receptor blockade with 8-cyclopentyl-1,3-dipropylxanthine, suggesting that caffeine attenuates MPTP toxicity by A(2A) receptor blockade. These data establish a potential neural basis for the inverse association of caffeine with the development of PD, and they enhance the potential of A(2A) antagonists as a novel treatment for this neurodegenerative disease.

Selective inhibition of MAO-B through chronic low-dose (R)-deprenyl treatment in C57BL/6 mice has no effect on basal neostriatal dopamine levels.

C. Thiffault, L. Lamarre-Théroux, R. Quirion, and J. Poirier (1997, Mol. Brain Res. 44: 238-244) recently reported that chronic treatment of young (12 week old) C57BL/6 mice with (R)-deprenyl, a mechanism-based inactivator of monoamine oxidase B (MAO-B), leads to a more than fourfold increase in neostriatal dopamine levels. Such an effect could complicate the interpretation of results obtained from mechanistic studies designed to evaluate the putative neuroprotective effects of (R)-deprenyl in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned mice. In contrast to the results of Thiffault et al., we have found that neostriatal dopamine levels in mature (32 week old) C57BL/6 mice were unaltered by chronic (R)-deprenyl treatment even though brain monoamine oxidase B activity was reduced by more than 80%. Neostriatal dopamine levels also were unaltered in both young and mature mice when the (R)-deprenyl treatment period was doubled compared to that reported by Thiffault et al. Consequently, studies on the putative neuroprotective properties of (R)-deprenyl in MPTP-lesioned mice are unlikely to be complicated by the possibility that inhibition of MAO-B alone will lead to an increase in neostriatal dopamine levels.

Study of the electrospray ionization mass spectrometry of the proton pump inhibiting drug Omeprazole.

A detailed analysis of the product ion spectrum generated from the protonated molecule under ESI-MS/MS conditions using a triple quadrupole mass spectrometer is reported for the gastrointestinal proton pump inhibitor Omeprazole. Unambiguous molecular composition data of the fragment ions were obtained with the aid of regioselectively 14C-, 34S- and 18O-labeled analogs. Attempts have been made to provide rational pathways for the formation of the fragment ions from four protonated omeprazole species. These results will facilitate the characterization of the complex metabolic fate of Omeprazole in humans, which involves the excretion of at least 50 metabolites.

Analysis of citrulline in rat brain tissue after perfusion with haloperidol by liquid chromatography-mass spectrometry.

We have investigated the potential of high-performance liquid chromatography (HPLC) coupled to mass spectrometry (MS) to determine enrichments of citrulline as a marker for in vivo nitric oxide (NO) production in brain tissue. The analysis of citrulline as the butyl ester derivative was evaluated using two types of ionization: electron spray ionization (ESI) and atmospheric pressure chemical ionization (APCI). APCI-MS appeared to be more suitable for determination of citrulline than ESI-MS, because the ion intensity of the protonated molecule ion [M+H]+, m/z 232, of citrulline in the former was about twelve times higher than in the latter. The chromatography was carried out on a reversed C8 column with the mobile phase consisting of 15% acetonitrile: 85% H2O: 0.2% acetic acid (v/v). The calibration curve had good linearity within the concentration range investigated (5 ng to 500 ng/ml). The limit of determination was estimated to be ca. 1 ng/ml of standard solution. The method was applied to the analysis of citrulline in the brain dialysate obtained from rat after perfusion of the striatum with haloperidol (HP, 0.1 mM). It is concluded that APCI-MS in combination with HPLC can be successfully applied to determination of citrulline in brain tissue, thus providing a useful tool for assessment of in vivo NO production.

Metabolic defects caused by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and by HPTP (the tetrahydropyridinyl analog of haloperidol), in rats.

The results of previous studies in the baboon have suggested that HPTP, the tetrahydropyridinyl analog of haloperidol causes a urinary biochemical marker profile similar to those seen in humans suffering from inborn errors of mitochondrial respiration. In order to identify a possible relationship between compromised cellular energy production and neuronal damage we now have compared the urinary profiles of rats treated with the pro-neurotoxin, MPTP as well as with HPTP. Significantly increased urinary excretion of lactic acid and 2-ethylhydracrylic acid in MPTP and HPTP treated rats was observed, indicating that both MPTP and HPTP and/or their respective metabolites cause mitochondrial inhibition in the rat.

Studies on the in vivo biotransformation of the tobacco alkaloid beta-nicotyrine.

This paper reports the results of studies on the in vivo metabolic fate of the tobacco alkaloid 1-methyl-2-(3-pyridinyl)pyrrole (beta-nicotyrine) in New Zealand white rabbits. Two previously characterized metabolites, 5-hydroxy-1-methyl-5-(3-pyridinyl)-2-pyrrolidinone (5-hydroxycotinine) and 2-hydroxy-1-methyl-5-(3-pyridinyl)-3-pyrrolin-2-one, were present in low concentrations in the urine of the treated animals. The major urinary metabolite of beta-nicotyrine was identified as cis-3'-hydroxy-1-methyl-5-(3-pyridinyl)-2-pyrrolidinone (cis-3'-hydroxycotinine), the diastereoisomer of the major urinary metabolite of (S)-nicotine. The pathway leading to cis-3'-hydroxycotinine is proposed to proceed via autoxidation of 2-hydroxy-1-methyl-5-(3-pyridinyl)pyrrole, a postulated cytochrome P450-generated metabolite of beta-nicotyrine, followed by reduction of the carbon-carbon double bond present in the resulting 3-hydroxy-3-pyrrolin-2-one species. This proposal is supported by the in vivo biotransformation of 2-acetoxy-1-methyl-5-(3-pyridinyl)pyrrole, a latent form of the putative hydroxypyrrole intermediate, to cis-3'-hydroxycotinine. The in vivo conversion of 5-hydroxy-1-methyl-5-(3-pyridinyl)-3-pyrrolin-2-one to 5-hydroxycotinine is offered as evidence that supports the proposed reduction step.

Isolation and characterization of a monoamine oxidase inhibitor from tobacco leaves.

Recent positron emission tomography imaging studies have demonstrated a significant decrease in both monoamine oxidase A and B (MAO-A and MAO-B) activities in the brains of smokers. Normal levels of activity are observed in former smokers, suggesting the presence of one or more compounds in tobacco smoke that may inhibit these enzymes. In this paper, we report the results of efforts to identify compounds present in flue-cured tobacco leaves that inhibit MAO. The isolation procedure was guided by estimating the inhibitory properties of tobacco leaf extracts on the liver mitochondrial MAO-B-catalyzed oxidation of 1-methyl-4-(1-methylpyrrol-2-yl)-1,2,3, 6-tetrahydropyridine to the corresponding dihydropyridinium metabolite. Fractionation of extracts from flue-cured tobacco leaves led to the isolation of a competitive inhibitor of human MAO-A (K(i) = 3 microM) and MAO-B (K(i) = 6 microM), the structure of which could be assigned by classical spectroscopic analysis and confirmed by synthesis. This information may help to provide insights into some aspects of the pharmacology and toxicology of tobacco products.

Melatonin fails to protect against long-term MPTP-induced dopamine depletion in mouse striatum.

Several laboratories recently have reported that melatonin may possess neuroprotective properties. The present paper presents the results of our studies on the long term in vivo neuroprotective effects of melatonin in a well-defined neurotoxicity model using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the C57BL/6 mouse. MPTP is bioactivated by brain monoamine oxidase B (MAO-B) to its neurotoxic pyridinium metabolite 1-methyl-4-phenylpyridinium (MPP(+)) which destroys dopaminergic nerve terminals leading to the depletion of neostriatal dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC). Our initial study compared striatal DA and DOPAC levels in MPTP-only-treated animals and animals treated with melatonin 30 min prior to and 3 times hourly post-MPTP. DA/DOPAC levels measured 7 days after MPTP were similar in both groups. A second study was designed to address the possibility that melatonin cleared from the brain prior to MPP(+). Animals, that had been administered the same regimen of melatonin as in the first study plus a fourth post-MPTP melatonin dose, were maintained on melatonin in drinking water until 5 days post-MPTP. Striatal DA/DOPAC levels of these melatonin-plus-MPTP treated animals also were the same as the MPTP-only-treated animals. In vitro studies confirmed that melatonin is not an inhibitor of MAO-B. These data demonstrate that melatonin does not have any significant protective effects against the long-term striatal DA and DOPAC depletion induced by MPTP in the C57BL/6 mouse.

Neuroprotection by (R)-deprenyl and 7-nitroindazole in the MPTP C57BL/6 mouse model of neurotoxicity.

The neurodegenerative properties of the parkinsonian inducing agent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) are thought to result from inhibition of complex I of the mitochondrial respiratory chain by the monoamine oxidase-B (MAO-B) generated 1-methyl-4-phenylpyridinium metabolite MPP+. 7-Nitroindazole (7-NI) both a reversible MAO-B inhibitor and a neuronal nitric oxide synthase (nNOS) inhibitor, and (R)-deprenyl a potent MAO-B inactivator, provide neuroprotection in the C57BL/6 mouse model of MPTP neurotoxicity. The results reported here demonstrate the complexities of the effects of 7-NI in this model and examine the possibility of other mechanisms of neuroprotection by (R)-deprenyl.

Species-dependent differences in monoamine oxidase A and B-catalyzed oxidation of various C4 substituted 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridinyl derivatives.

In an attempt to provide a better understanding of the scope and limitations of animal models used in some drug development programs and to further our understanding of potential metabolic bioactivation reactions, we have undertaken studies to profile the monoamine oxidase A and B (MAO-A and -B, respectively) activities in liver and brain mitochondrial preparations obtained from a variety of species using a series of 1-methyl-4-aryl-1,2,3, 6-tetrahydropyridinyl substrates. Mitochondrial preparations were incubated with substrates at 37 degrees C in the presence or absence of clorgyline, (R)-deprenyl, or a mixture of these two propargylamines to inhibit MAO-A, MAO-B, or both enzymes. The rates of formation of the corresponding dihydropyridinium metabolites were estimated spectrophotometrically. MAO-B was found to be the principal enzyme present in all tissues. Human liver displayed more MAO-A activity than the liver of any other species studied; subhuman primates displayed little or no detectable MAO-A activity. The properties of the preparations from rat liver were most similar to those from human liver with respect to the MAO-A/MAO-B ratios and the kinetic parameters of the four substrates used to profile enzymatic activity. The kinetic properties of mitochondrial preparations from bovine liver, a commonly used source of purified MAO-B preparations, were consistently different from all of the other species studied. The mitochondrial preparations from rabbit brain and liver also were unusual in that they displayed relatively low MAO activities. Additionally, these enzyme activities were considerably less susceptible to inhibition by clorgyline and (R)-deprenyl. Finally, an exceptionally low MAO-B liver/brain V(max)/K(m) ratio was observed with the mitochondria obtained from the C57BL/6 mouse, an effect that may contribute to the susceptibility of this strain to the toxic effects of the parkinsonian-inducing neurotoxin 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine.

p-Fluorophenylglycine in the urine of baboons treated with HPTP, the tetrahydropyridine analog of haloperidol.

We report the presence of p-fluorophenylglycine (p-FPG) in the urine of six baboons treated with HPTP, the tetrahydropyridine dehydration product of haloperidol (HP). Oxidative N-dealkylation, the major metabolic pathway of HP, gives rise to 3-(4-fluorobenzoyl)propionic acid (p-FBPA). Subsequent beta-oxidation of p-FBPA produces p-fluorophenylacetic acid (p-FPA). The presence of p-FPA argues for the formation also of p-fluorophenylglyoxylic acid (p-FPGA) derived from beta-oxidation of p-FBPA. Plasma aminotransferases should convert p-FPGA to p-FPG. The presence of p-FPG in these animals suggest the presence of phenylglycine aminotransferases in the baboon and possibly also in other primates, including the human. Reports by other authors found that treatment with alpha-phenylglycine (alpha-PG), an "unnatural" amino acid, leads to striatal dopamine (DA) depletion in rabbits--an effect explained on the basis of alpha-PG competing with DA for the neuronal vesicular storage sites. We performed in vitro DA release assays in mouse striatal synaptosomal preparations but found that neither alpha-PG nor p-FPG released any DA. It therefore remains unclear whether p-FPG may be a contributing factor to neurologic side-effects such as tardive dyskinesia (TD) found in patients after long-term HP treatment.

Clinical and neuropathological abnormalities in baboons treated with HPTP, the tetrahydropyridine analog of haloperidol.

Tardive dyskinesia (TD) is relatively common among psychiatric patients on maintenance therapy with typical neuroleptics and persists in more than 20% even after withdrawal of the medication. Such persistence suggests an underlying pathology due to neurotoxicity. We present evidence for such a neurotoxic mechanism in a baboon model of TD. Four baboons were treated chronically with the dehydration product of haloperidol, 4-(4-chlorophenyl)-1-[4-(4-fluorophenyl)-4-oxobutyl]-1,2,3,6- tetrahydropyridine (HPTP), which is metabolized, similarly to haloperidol, to two neurotoxic pyridinium species. The animals developed orofacial dyskinesia which persisted after HPTP was ceased. Serial sections of the entire brain from the four treated animals and four vehicle-treated controls revealed volume loss in the basal forebrain and hypothalamus. Histological evaluation demonstrated a reduction in the density of magnocellular neurons in the anterior region of the nucleus basalis of Meynert (NbM). We speculate that the loss of these NbM neurons may be associated with the persistent orofacial dyskinesia observed in the HPTP-treated animals. These findings may contribute to a better understanding of neuroleptic-induced TD.